An intermediate form of ADP-F-actin

With yeast actin, contrary to other actins, filament formation, ATP hydrolysis, and P-i release are concurrent at low actin concentrations, the condition usually employed to assess actin polymerization. This observation leads to a question concerning the conformation of the filament barbed end that might be recognized by specific actin-binding proteins. To try to detect possible new actin polymer conformations that might be intermediate in the pathway leading to mature F-actin, we monitored the change in intrinsic tryptophan fluorescence of yeast and muscle actins polymerized at pH 6 to accelerate the rate of filament formation. This allowed temporal resolution of the P-i release process from the slower process of polymerization. With both actins, we detected a biphasic instead of the usual monophasic fluorescence change, a rapid decrease that tracks with filament formation followed by a slower rebound ( the second phase). This second phase postpolymerization conformational change requires P-i release and occurs nearly coincident with its release. The addition of P-i causes this second phase response to disappear, and the inclusion of P-i during polymerization prevents its appearance. At pH 7.5, with higher yeast actin concentrations to accelerate polymerization, a two-phase fluorescence change is also observed. In this case, the second phase change lags substantially behind P-i release. P-i release could also be resolved from polymer formation. V159N yeast actin, hypothesized previously as remaining in a postpolymerization ATP-like state, exhibits the same two-phase intrinsic tryptophan fluorescence behavior as wild-type yeast actin. Together, these observations demonstrate the presence of an intermediate filament state between ADP-P-i and mature ADP-F-actin.